Process for forming a polymer layer with a uniform thickness on the surface of a solid support, solid support obtained and its applications

ABSTRACT

The present invention relates to a process for forming a polymer layer with a uniform thickness on the surface of a solid support, to the solid support obtained using this process, and to its applications.

[0001] The present invention relates to a process for forming a polymerlayer with a uniform thickness on the surface of a solid support, to thesolid support obtained using this process, and to its applications.

[0002] At the present time, there is a great interest in themultiparameter analytical tools known as DNA chips. These tools arededicated to the analysis of biological macromolecules such as, forexample, nucleic acid molecules and proteins.

[0003] They make it possible, by means of a polymer layer bonded to atleast one surface of a solid support, to fix these biologicalmacromolecules by means of specific functional groups. The moleculesthus immobilized can then be subjected to various qualitative andquantitative analytical techniques or can also serve as probes, for thepurpose of fixing other biological molecules of interest, especially inthe case of the hybridization of complementary DNA strands.

[0004] At the present time, the deposition of polymers, and inparticular of conducting polymers combined with specific (biological orchemical) functional groups for the purpose of fixing biologicalmacromolecules, is carried out in various ways, including chemical orelectrochemical deposition.

[0005] The polymer layers of the systems currently available on themarket are of greater or lesser thickness depending on the desiredapplications.

[0006] The deposited polymer layers associated with the fixing ofbiological elements are described as a succession of links of a chainformed by one or more different monomers, some of these differentmonomers themselves carrying biological elements.

[0007] As an illustration, this kind of construction may be shownschematically by the following structure:

[0008] in which A and B represent the monomers of the polymer and BErepresents a biological element carried directly by the monomers Aand/or B.

[0009] In the particular case of conducting polymers, the polymerizationis carried out electrochemically. During the polymerization process, theconducting polymer chain grows by successive insertion of the variousmonomers. Statistically, the monomers carrying biological elements arerandomly fixed to the chain as it grows in length. Since the length ofthe chain is not controlled, it continues to grow until thepolymerization process is stopped, this usually being caused, over time,by reducing the deposition potential or else by the exhaustion of themonomers. This technique does not allow the length of the chains to beeasily controlled and consequently results in polymer layers beingobtained which are nonuniform in terms of thickness.

[0010] Moreover, when the polymerization is carried out chemically, achemical oxidizing agent is, for example, mixed with the monomers, atthe time of use, in order to initiate the polymerization process(oxidative polymerization). In this case, it is only the time for whichthe various monomers and the chemical oxidizing agent are left incontact with each other, or the amounts of monomers initially present,which allows the final length of the polymer chains to be varied. Thispolymerization technique therefore allows easy control neither of thefinal thickness of the polymer layer formed, nor of the amounts of anybiological elements fixed to the chains thus formed.

[0011] Moreover, since the biological elements are more accessible atthe surface of the polymer layers than within this layer, many workershave sought to reduce the thicknesses of the polymer layers bydepositing them rapidly (short deposition time, generally less than 1second).

[0012] Such rapid deposition requires great control of the parametersinvolved during preparation of the polymer layer (polymerization time,deposition potential in the particular case of electrochemicaldeposition, surface cleaning, etc.). Rapid deposition, although allowingthin polymer layers to be obtained, is therefore difficult to implementand requires very precise operating conditions.

[0013] Moreover, when it is desired to polymerize several sitessimultaneously, the deposition conditions mean that it is extremelydifficult to achieve uniform deposition thicknesses at all the sites.This is because the physico-chemical conditions of the surface of thesite to be polymerized affect the start of deposition, with theconsequence that the thickness of the various polymer layers obtained onthe various sites to be polymerized is nonuniform. This is typically thecase when producing biochips of the MICAM® type, such as those describedfor example in patent application FR-A-2 781 886 and in the article byM. P. Caillat, “Sensors and Actuators”, B-Chimie, 1999, 61, 154-162.

[0014] It is therefore to remedy all these drawbacks and to provide aprocess for obtaining solid supports, at least one of the surfaces ofwhich is covered with a uniform polymeric layer of small thickness, thatthe inventors have developed what forms the subject matter of thepresent invention.

[0015] The inventors have in particular demonstrated, surprisingly andunexpectedly, that it is possible to prepare such supports using amixture of various monomers, the said mixture consisting, to at least50%, of monomers having a chemical functional group capable of blockingthe polymerization reaction.

[0016] The first subject of the present invention is therefore a processfor forming a polymeric layer with a uniform thickness on at least partof one of the surfaces of a solid support, wherein said polymeric layeris obtained by copolymerization of a monomer solution containingmonomers carrying a functional group capable of stopping thecopolymerization reaction, and in which solution the amount of monomerscarrying said functional group represents at least 50% of the totalamount of monomers present within said solution.

[0017] The inventors have in fact demonstrated that the processaccording to the invention makes it possible for the deposition of themonomers, and thereby the growth of the polymer chains, to beself-regulated by successively stopping the growth of the chainsundergoing polymerization. This makes it possible to greatly reduce therate of chain growth and to obtain more flexible conditions fordeposition at the sites that it is desired to cover. Surprisingly, thisprocess then makes it possible to obtain remarkably uniform thin polymerlayers.

[0018] In addition, when it is desired for the polymerization to becarried out at several sites simultaneously, for example in the case ofbiochips possessing several thousands of different sites, the initiationof polymerization may differ between two sites because of thephysico-chemical conditions of the surfaces. In this case, the processaccording to the invention makes it possible to retard the growth of thelayer by successive stopping of the growing chains and to reduce thedifference owing to delayed initiation between two sites as the initialdeposit continues to grow. In the end, the difference in thicknessbetween the various sites is reduced and the biochip has uniform polymerthicknesses at each of its various sites.

[0019] The process according to the invention also allows the amounts ofbiological elements fixed to a polymer chain to be limited andcontrolled.

[0020] According to an advantageous embodiment of the invention, theamount of monomers carrying said functional group preferably representsat least 60%, and even more preferably 60 to 95%, of the total amount ofmonomers present in said solution.

[0021] The process according to the invention makes it possible tocontrol the growth of the polymer layer, the thickness of which istherefore more uniform. More specifically, the process according to theinvention makes it possible to reduce the rate of formation of thepolymer layer by a factor of about 10. The standard deviation (E) of thethickness measurement (made on polymer layers whose thickness may varybetween a few nanometers and a few tens of nanometers) may be calculatedfrom the following equation:$E = \sqrt{\frac{1}{n}{\sum( {X_{i} - X_{m}} )^{2}}}$

[0022] in which n represents the amount of measurements, X₁ representsthe thickness value in ångstroms at the measurement point and X_(m)represents the mean thickness value.

[0023] This standard deviation E goes from 90 for a polymeric layerobtained by using only unfunctionalized monomers to 9 only if one uses amonomer mixture containing about 85% of monomers functionalized by afunctional group capable of stopping the copolymerization reaction withrespect to the total amount of monomers.

[0024] According to the invention, the actual nature of the monomers isnot critical.

[0025] The monomers that can be used in the process according to theinvention are in fact chosen depending on the type of applicationenvisioned and especially on the types of macromolecules that it isdesired to fix.

[0026] These monomers may be especially chosen from pyrroles, anilines,thiophenes and azulenes.

[0027] The copolymerization reaction may be initiated, depending on thenature of the monomers, either by a chemical oxidizing agent or by anelectrical signal (in electropolymerization) in the case of conductingmonomers.

[0028] Among chemical oxidizing agents, mention may especially he madeof ferric chloride, cupric chloride, molybdenum (V) chloride andruthenium trichloride.

[0029] According to a preferred embodiment of the invention, saidmonomers are chosen from pyrrole monomers and the copolymerizationreaction is an electropolymerization reaction.

[0030] Among functional groups capable of stopping the copolymerizationreaction, mention may especially be made of aldehyde, nitrile, ester,alkyl and aryl functional groups.

[0031] Advantageously, said functional group is preferably chosen fromthose allowing attachment of a biological or biochemical elementcontaining, for example, an amine functional group, such as for examplea DNA strand, a biotin, a streptavidin or an antibody.

[0032] As an example, and when the monomer is a pyrrole carrying analdehyde functional group, the attachment of the biological element willbe able to take place directly on the functional group capable ofstopping the polymerization reaction, according to the followingreaction:

[0033] in which R represents a biological or biochemical element.

[0034] Thus, a solid support will be obtained which has a polymericlayer in which the biological or biochemical element is fixed to the endof a chain according to the following formula:

[0035] in which R represents a biological or biochemical element and Scorresponds to the surface of a solid support.

[0036] This process therefore also allows solid supports to be obtainedin which the polymer layer is particularly uniform and whose solidsupports offer the further advantage of having biological or biochemicalelements that are very accessible. This accessibility is particularlybeneficial if it is desired to carry out hybridization reactions on DNAstrands.

[0037] The solid supports that can be used according to the inventionare preferably chosen from metal supports, such as gold supports, orglass or plastic supports.

[0038] The subject of the invention is also the solid support obtainedby implementing the process described above. This solid supportincludes, on at least part of one of its surfaces, a polymer layer whosethickness is preferably between about a few nanometers and a few tens ofnanometers.

[0039] The subject of the invention is also the use of at least onesolid support according to the invention, for isolating, separatingand/or analyzing biological and biochemical elements. Thus, oneapplication of the invention is in the production of improvedMICAM®-type biological chips, but also biological chips whose solidsupport is made of glass or plastic.

[0040] Apart from the above arrangements, the invention also comprisesother arrangements which will emerge from the description which follows,this referring to an example of a measurement of the rate of formationof a polypyrrole layer on the surface of a solid support as a functionof the pyrrole/(pyrrole carrying an aldehyde functional group) ratio, toan example of the measurement of the thickness and the uniformity of apolymeric layer on the surface of a solid support as a function of thepyrrole/(pyrrole carrying an aldehyde functional group) ratio, and tothe appended figures in which:

[0041]FIG. 1 shows the rate of formation of the polymer layer (as apercentage of the initial rate) as a function of the percentage ofpyrrole-2-carboxaldehyde monomer; and

[0042]FIG. 2 shows the standard deviation E of the thickness measurementas a function of the decreasing percentage of pyrrole-2-carboxaldehydepresent in each monomer solution.

EXAMPLE 1 Measurement of the Rate of Formation of a Polypyrrole-TypePolymer Layer as a Function of the Percentage ofpyrrole-2-carboxaldehyde Monomers Present Within apyrrole/pyrrole-2-carboxaldehyde Monomer Mixture

[0043] 1) Equipment and Method

[0044] Nine pyrrole/pyrrole-2-carboxaldehyde (Aldrich) monomer solutionscomprising from 10 to 90% of pyrrole-2-carboxaldehyde monomer relativeto the total amount of pyrrole/pyrrole-2-carboxaldehyde monomers (with a1 molar concentration in acetonitrile) were prepared.

[0045] Two monomer solutions (with a 1 molar concentration inacetonitrile) consisting, respectively, of only pyrrole monomers and ofonly pyrrole-2-carboxaldehyde monomers were also prepared.

[0046] The various monomer solutions thus prepared were deposited ongold terminals all having the same size. The deposition potential wasset at 1.1 V (saturated calomel electrode). For each solution,deposition was carried out until a charge of 5 mC was obtained. The timeneeded to reach this charge was recorded. The mean deposition currentwas calculated by dividing the total charge by the deposition time. Thepolymerization reaction was initiated electrochemically. The supportelectrolyte used was 0.1 M aqueous LiClO₄. The rate of formation of thepolymeric layer is given by the mean deposition current.

[0047] 2) Results

[0048] By comparing the various mean currents obtained with one another,and by setting the maximum rate obtained at 100%, a curve showing therate of formation of the polymer layer as a function of the percentageof pyrrole-2-carboxaldehyde monomer present in each monomer solution wasobtained (FIG. 1).

[0049] These results show that a solution of pyrrole monomers containingat least 50% in number of pyrrole-2-carboxaldehyde monomer results in areduction of about 10% in the rate of formation of the polymer layer.When the percentage of pyrrole-2-carboxaldehyde monomer is between 60and 70%, the rate of formation of the polymer is reduced of about 50%.

EXAMPLE 2 Measurement of the Uniformity of a Polypyrrole-Type PolymerLayer as a Function of the Percentage of pyrrole-2-carboxaldehydeMonomer Present Within a pyrrole/pyrrole-2-carboxaldehyde MonomerMixture

[0050] 1) Equipment and Method

[0051] Four pyrrole/pyrrole-2-carboxaldehyde (Aldrich) monomer solutionscomprising from 46 to 86% (46%; 66%; 78% and 86%) ofpyrrole-2-carboxaldehyde monomer relative to the total amount ofpyrrole/pyrrole-2-carboxaldehyde monomers (with a 1 molar concentrationin acetonitrile) were prepared.

[0052] Two monomer solutions (with a 1 molar concentration inacetonitrile) consisting, respectively, of only pyrrole monomers and ofonly pyrrole-2-carboxaldehyde monomers were also prepared.

[0053] The various monomer solutions thus prepared were depositedaccording to the method described above in Example 1.

[0054] For each solution, deposition was carried out until a charge of15 mC was obtained.

[0055] The thickness and the uniformity of the deposited layer were thenmeasured by means of an optical interferometer of the NANOSPEC® brand.The refractive index of the layer was set arbitrarily at 1.7.

[0056] Each deposited layer was measured at five points.

[0057] The uniformity of the deposited layer is characterized by thestandard deviation (E) of the thickness measurements obtained.

[0058] The results obtained are plotted in FIG. 2, which shows thestandard deviation E of the thickness measurement as a function of thedecreasing percentage of pyrrole-2-carboxaldehyde present in eachmonomer solution. These results show that the standard deviation E isdecreased by a factor of about 2 when the monomer mixture used contains,in accordance with the invention, 66% of monomers carrying a functioncapable of stopping the copolymerization reaction. These results alsoshow that the standard deviation E goes from about 95 to only about 9when the percentage of pyrrole-2-carboxaldehyde monomer goes from 0% to85%.

[0059] These results are indicative of the very great uniformity of thepolymeric layer obtained by implementing the process according to thepresent invention.

1. A process for forming a polymeric layer with a uniform thickness onat least part of one of the surfaces of a solid support, wherein saidpolymeric layer is obtained by copolymerization of a monomer solutioncontaining monomers carrying a functional group capable of stopping thecopolymerization reaction, and in which solution the amount of monomerscarrying said functional group represents at least 50% of the totalamount of monomers present within said solution.
 2. The process asclaimed in claim 1, wherein the amount of monomers carrying saidfunctional group represents at least 60% of the total amount of monomerspresent in said solution.
 3. The process as claimed in claim 2, whereinthe amount of monomers carrying said functional group represents from 60to 95% of the total amount of monomers present in said solution.
 4. Theprocess as claimed in any one of the preceding claims, wherein themonomers are chosen from pyrroles, anilines, thiophenes and azulenes. 5.The process as claimed in claim 4, wherein the monomers are chosen frompyrroles and the copolymerization reaction is an electropolymerizationreaction.
 6. The process as claimed in any one of the preceding claims,wherein the functional groups capable of stopping the copolymerizationreaction are chosen from aldehyde, nitrile, ester, alkyl and arylfunctional groups.
 7. A solid support, which is obtained using theprocess as defined in any one of claims 1 to
 6. 8. The use of at leastone solid support as defined in claim 7 for isolating, separating and/oranalyzing biological and biochemical elements.
 9. The use of at leastone solid support as defined in claim 7 for producing biological chips.